Data from: A sequel to Sanger: amplicon sequencing that scales

Although high-throughput sequencers (HTS) have largely displaced their Sanger counterparts, the short read lengths and high error rates of most platforms constrain their utility for amplicon sequencing. The present study tests the capacity of single molecule, real-time (SMRT) sequencing implemented on the SEQUEL platform to overcome these limitations, employing 658 bp amplicons of the mitochondrial cytochrome c oxidase I gene as a model system. By examining templates from more than 5,000 species and 20,000 specimens, the performance of SMRT sequencing was tested with amplicons showing wide variation in GC composition and varied sequence attributes. SMRT and Sanger sequences were very similar, but SMRT sequencing provided more complete coverage, especially for amplicons with homopolymer tracts. Because it can characterize amplicon pools from 10,000 DNA extracts in a single run, the SEQUEL reduces costs 40-fold from Sanger analysis. Reflecting the capacity of each instrument to recover sequences from more than five million DNA extracts a year, this platform facilitates massive amplicon characterization.

尽管高通量测序仪（high-throughput sequencers, HTS）已在很大程度上取代了桑格（Sanger）测序仪，但多数平台存在读长偏短、错误率较高的问题，限制了其在扩增子测序中的应用潜力。本研究针对SEQUEL平台搭载的单分子实时测序（single molecule, real-time, SMRT）技术克服上述局限的能力展开测试，以线粒体细胞色素c氧化酶I基因的658 bp扩增子作为模型系统。研究通过检测超过5000个物种、20000份标本的模板，对GC组成和序列属性存在广泛差异的扩增子开展SMRT测序性能评估。结果显示，SMRT测序与桑格测序结果高度相似，但SMRT测序可提供更完整的覆盖度，尤其针对带有同源多聚体区域的扩增子。由于单次运行即可完成10000份DNA提取物对应的扩增子混合池的序列表征，SEQUEL平台的测序成本较桑格分析降低了40倍。考虑到该仪器每年可从超过500万份DNA提取物中获取序列数据，该平台能够实现大规模扩增子测序表征。